Project Summary The nasal cavity is constantly exposed to airborne particles, some of which are potentially damaging to the respiratory tract. Noxious particles are trapped in the mucosal layer and are expelled through mucociliary clearance. Some particles trigger a local, protective inflammatory response through stimulation of solitary chemosensory cells that results in activation of peptidergic nerve fibers. Solitary chemosensory cells respond to numerous airborne irritants through receptors on their apical microvillar processes. The range of substances these cells are known to detect include bitter substances, homoserine lactones excreted by bacteria, and some odorous irritants. While these cells are generally protective, chronic stimulation could contribute to the etiology of airway disorders including chronic rhinosinusitis and conductive smell loss. The current proposal seeks to leverage innovative technologies to explore how this single population of cells is able to detect a wide variety of airborne irritants. First, single cell RNA-sequencing will be used to examine the heterogeneity of solitary chemosensory cells. The types of chemoreceptors on individual cells will be investigated and statistical methods will be used to cluster certain populations of cells based on the transcripts they contain. These results will help define specific signaling mechanisms by which certain stimuli could activate different classes of solitary chemosensory cells and signal to the nervous system or surrounding tissue. In the second part of this proposal, the distribution and innervation of solitary chemosensory cells in the entire nasal cavity will be explored using optical clearing methods and light sheet microscopy. The use of light sheet microscopy on optically cleared tissues prevents the need to physically section tissue samples for immunocytochemistry. By combining these methods, three dimensional reconstructions of large volumes of tissues can be generated to ascertain accurate spatial information. Specifically, subpopulations of solitary chemosensory cells and nerve fibers will be visualized using antibodies or RNA-probes. These experiments will address whether certain populations of solitary chemosensory cells are spatially segregated in the nasal cavity and whether certain populations are more or less likely to be innervated by peptidergic nerve fibers. The results from this study will be beneficial to chemosensory and respiratory system scientists. Currently, solitary chemosensory cells are usually referred to as a homogenous population of chemosensors in the nasal cavity that trigger inflammatory responses through a single mechanism; however, the results from this study will reshape current knowledge of solitary chemosensory cells by defining subtypes and examining their distribution patterns within the nose. These results will give insight to ways that specific stimuli could activate certain classes of solitary chemosensory cells and communicate this information to the nervous system or surrounding tissues.